A very fast and easy method for the size-selective removal of smaller DNA from larger fragments. By adjusting the PEG and MgCl<sub>2</sub> concentration the range of precipitated DNA fragments can be adjusted.

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A very fast and easy method for the size-selective removal of smaller DNA from larger fragments. By adjusting the PEG concentration the range of precipitated DNA fragments can be adjusted.

==Materials==

==Materials==

===Reagents===

===Reagents===

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*DNA to be separated

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*DNA to be separated (e.g. PCR reaction mixture)

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*30% (w/v) PEG 8000/30 mM MgCl<sub>2</sub> (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA)

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*30% (w/v) PEG 8000/30 mM MgCl<sub>2</sub> (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA. The concentration used here will remove DNA fragments with less than 300bp)

*TE Buffer, pH 8.0 (10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)

*TE Buffer, pH 8.0 (10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)

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==Critical steps==

==Critical steps==

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*Before centrifugation mark the tubes in order to know where the pellet will be expected afterwards, as the pellet will be (nearly) invisible

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==Other PEG Concentrations and Approximate Size Exclusion==

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% PEG -- Fragments Excluded <br>

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*10% -- <300bp

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*6% -- <500bp

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*5% -- <700bp

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*4% -- <1kb <br>

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Notes:

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**Yield decreases considerably when <10% PEG is used

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**Always use 10mM final concentration of MgCl<sub>2</sub>

==Troubleshooting==

==Troubleshooting==

==Notes==

==Notes==

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*This protocol is being mentioned in the manual for the Gateway recombinational cloning system based on published methods.<cite>Website Paithankar Lis</cite>

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==Acknowledgments==

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*Adjusting the PEG concentration can shift the threshold of the size of the precipitated DNA. The higher the final PEG concentration, the smaller the fragments that will be removed (which will stay in the supernantant).

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Acnkowledge any help you had in development, testing, writing this protocol.

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*If the sample volume is different, simply adjust the other volumes accordingly to end up with the same ratio.

==References==

==References==

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See [[OpenWetWare:Biblio]] for information on how to reference within a wiki.

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<biblio>

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#Website http://www.invitrogen.co.jp/focus/181027.pdf

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#Paithankar pmid=2030954

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#Lis pmid=6246357

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</biblio>

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==Specific Protocols==

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Add links to all the OWW protocols that have been used in making the consensus.

==Discussion==

==Discussion==

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Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories.

Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories.

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[[Category:Protocol]]

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==BioCoder version==

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Following is the Size selective DNA precipitation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]]

Curators

Kersten S. Rabe

Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently discussing the idea of protocol curators. Please contribute.

Abstract

A very fast and easy method for the size-selective removal of smaller DNA from larger fragments. By adjusting the PEG concentration the range of precipitated DNA fragments can be adjusted.

Materials

Reagents

DNA to be separated (e.g. PCR reaction mixture)

30% (w/v) PEG 8000/30 mM MgCl2 (concentration of PEG 8000 can be varied to shift the size of the percipitated DNA. The concentration used here will remove DNA fragments with less than 300bp)

TE Buffer, pH 8.0 (10 mM TRIS-HCl, 1 mM EDTA, pH 8.0)

Equipment

Centrifuge which can do up to 10.000 rcf (=g)

Appropriate tubes for the centrifuge

Pipettes

Vortexer

Procedure

Mix 50 μL of sample with 150 µL of TE

Add 100 µL of PEG/MgCl2

Vortex

Centrifuge 15 min at 10.000 rcf at roomtemperature

Carefully remove supernatant not to disturb the pellet, which will be invisible

Dissolve the pellet in a appropriate amount of buffer of choice

Critical steps

Before centrifugation mark the tubes in order to know where the pellet will be expected afterwards, as the pellet will be (nearly) invisible

Other PEG Concentrations and Approximate Size Exclusion

% PEG -- Fragments Excluded

10% -- <300bp

6% -- <500bp

5% -- <700bp

4% -- <1kb

Notes:

Yield decreases considerably when <10% PEG is used

Always use 10mM final concentration of MgCl2

Troubleshooting

Notes

This protocol is being mentioned in the manual for the Gateway recombinational cloning system based on published methods.[1, 2, 3]

Adjusting the PEG concentration can shift the threshold of the size of the precipitated DNA. The higher the final PEG concentration, the smaller the fragments that will be removed (which will stay in the supernantant).

If the sample volume is different, simply adjust the other volumes accordingly to end up with the same ratio.

Discussion

Tag this page with categories to allow easier indexing and searching. See Categories for information on existing categories.

BioCoder version

Following is the Size selective DNA precipitation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi